Stationary capillary electrophoresis system

ABSTRACT

A system and method for capillary electrophoresis are provided for analyzing a macromolecule prepared from a complex liquid mixture. In particular applications, methods and apparatus are provided for separating and analyzing a solution containing a denatured macromolecule by employing a stationary capillary electrophoresis apparatus. An apparatus for capillary electrophoresis includes an inlet chamber and a capillary electrophoresis column. One end of the column is fixed at the interior of the inlet chamber. The column has a length of at least about 20 centimeters. Also included is a liquid source adapted for automatic control. The liquid source supplies a liquid sample through an input valve into the inlet chamber so that the sample is in fluid communication with the end of the column. A method for capillary electrophoresis includes automatically supplying the liquid sample to the apparatus.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/601,181, filed Jun. 20, 2003. The entire teachings of the aboveapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Analysis of macromolecules in complex mixtures is challenging in manychemical and biochemical processes. For example, the analysis of amacromolecule product, e.g., a protein, typically involves firstpreparing a sample of a macromolecule from a complex mixture foranalysis. FIG. 1 depicts an example of a macromolecule preparationprocess 100, which involves taking a sample from a complex liquidmixture, e.g. a biofluid in a bioreactor 102, separating a macromolecule104 from other components in the mixture, and processing it to deliver aprepared macromolecule 104′ for analysis at analyzer 106.

Effective process control generally requires accurate and frequentsampling, yet sampling of an operating bioreactor is associated withnumerous problems, particularly contamination from sampling. Forexample, a bioreactor fluid typically contains, in addition to themacromolecule of interest, components such as salts, nutrients,proteins, peptides, cells, cell components, biopolymers such aspolysaccharides, and the like, all of which can confound analysis of thedesired products. Sampling can introduce, for example, foreign or wildbacteria into a bioreactor, which can compete with the process bacteriain the bioreactor fluid. Other contaminants, e.g., chemicalcontaminants, can affect the growth of the process bacteria and canconfound the analysis of process components in the bioreactor fluid.Contamination can also affect the sampling and analysis apparatus. Forexample, wild or process bacteria can colonize the sampling/analysissystem, or the system can accumulate other components form the biofluid,e.g., as salts, nutrients, proteins, peptides, cells, cell components,biopolymers such as polysaccharides, all of which can confound analysisof the desired products. Additionally, frequent sampling can lead tobuild-up of the molecule or molecules being analyzed, which can lead toinaccuracy.

In particular, the problem of “backflow”, i.e., liquidcross-contamination, is especially difficult when interfacing twofluidic systems. Simple valve interfaces are inadequate because valvestypically have crevices, joints, dead volume, and the like, wherecontaminants can lodge and accumulate, only to be released duringanother sample cycle. Additionally, valves can fail and allowundesirable contamination to occur before much measurable fluid hasleaked. More complex valved interfaces are known, but some are costlyand still suffer some of the problems of simple valve systems, whileother examples are unsuitable for high pressure systems. Needle/septainterfaces are known to avoid backflow but have issues with septalifetime, needle contamination during transfer, and are particularlytroublesome for frequent, automated sampling of larger volumes.Furthermore, septa replacement itself opens the system forcontamination.

FIG. 2 depicts typical steps that can be included in a macromoleculesample preparation process operating on a mixture 202. If themacromolecule is endogenous, i.e., is at least partly contained incells, an optional lysing step 204 opens the cells so that themacromolecule 104 can be separated. Separation step 206 separatesmacromolecule 104 from rough components 207 and fine components 213.Rough components 207 can include, for example, insoluble cells 208,cellular fragments 210, soluble molecules 212 which are larger thanmacromolecule 104, and the like. Fine components 213 can include salts214 and soluble molecules 215 that are smaller than macromolecule 104,and the like. The concentration of ions such as salts and hydrogen(i.e., pH) are adjusted in step 216. In step 218, the molecule can bedenatured, i.e., can be heated and/or combined with a denaturing agent220, producing prepared macromolecule 104′, which is typically at anincreased concentration compared to macromolecule 104.

The various steps used for protein preparation in the prior art involveseparation of components through labor intensive centrifugation ortime-intensive matrix chromatography. Matrix chromatography usesexpensive columns that can be prone to plugging when used with complexmixtures that include insoluble or precipitation-prone components.Centrifugation can be effective but can cause contamination problems asthere is no way to readily isolate a sample from the environment duringthe various sample transfers typically employed, and the size of thecentrifuge limits the amount of macromolecule that can be prepared atone time. Thus both methods are low throughput in terms of amount ofmacromolecule that can be prepared.

Additionally, both methods are low throughput in terms of the samplingfrequency, as the time from sample extraction from a complex bioreactormixture to analysis of the macromolecule can easily be four hours ormore. Such a slow analysis time leads to poor optimization of reactorprocesses, resulting in lowered yields, increased costs, increasedpurification demands, and increased amounts of potentially hazardousbiological waste. FIG. 3 depicts a hypothetical example comparing twosampling frequencies, wherein a lower sampling frequency versus time(squares) can miss details in the level of a desired macromoleculeversus time (solid line) in a reaction mixture, compared to a highersampling frequency (circles). For example, the lower sampling frequencycan miss the maximum macromolecular concentration 302 by measuring onlylower concentration 300.

Electrophoresis is an analytical technique commonly used to separatemolecular species, e.g., peptides, proteins, oligonucleotides, smallorganic molecules, and the like. The molecules, in a separation medium,e.g., a solution or a gel matrix, separate under an applied electricfield according to their electrophoretic mobility, which is related tothe charge on each molecule, its size, and the viscosity of theseparation medium.

FIG. 10 depicts the separation of a small molecule 1002 and a largemolecule 1004, each with the same net positive charge, and a smallnegatively charged molecule 1006. Application of electric field 1008causes differential motion of the charged molecules according to theirelectrophoretic mobilities, with cations 1002 and 1004 moving towardsthe cathode 1010. In the ideal case, the anions 1006 move to the anode1012, though experimentally a phenomenon known as electroosmotic flowcan reduce or reverse the anion to anode motion.

In capillary electrophoresis (CE), the separation is performed in acapillary tube having an internal diameter on the order of tens tohundreds of micrometers. In such small tubes the heat generated by theelectric field is easily dissipated, so that high electrical fields canbe used, leading to fast separations. FIG. 11 depicts a schematic of anelectrophoresis apparatus 1100. An inlet vessel 1102 and an outletvessel 1104 are connected by a capillary column 1106. The vessels andthe capillary contain a buffer with an appropriate electrolyte. Uponloading a sample containing the analyte of interest at the inlet vessel,an electric field provided by a high voltage power supply 1108 causesthe various molecules in the sample to separate, whereupon they can bedetected by a detector 1110.

While capillary electrophoresis is powerful and versatile, it issensitive to variations in acidity (pH), ionic strength, temperature,viscosity and other physical characteristics of the mixture, propertiesintrinsic to the analytes being studied, and contamination issues.Furthermore, small capillaries are physically fragile and are not suitedto high-throughput separations, being easily plugged from the manymacromolecules and debris in a complex mixture. In particular, rapidseparation and analysis of macromolecules from complex liquid mixtures,for example, during the analysis of proteins produced in a bioreactor,is especially challenging.

In one example of CE technology a fragile, small diameter capillary isrepeatedly applied by robotics to a series of distinct inlet vials. Therepetitive motion can easily break the CE column. Column replacementrequires time-consuming recalibration of the robotic motion. Anotherexample of CE technology employs microchannels etched into a glass chip.While this hardware is durable, the separation efficiency is limited bythe length of CE channel that can be fabricated on a chip. Attempts toextend the channel length by increasing channel density on a chipgenerally restrict high electric fields from use, increasing separationtime. Also, the throughput of this technique is limited. Furthermore,sample transfer as practiced in both the robotic capillary technique andthe chip technique expose the analytic solution to undesirableenvironmental contamination.

SUMMARY OF THE INVENTION

A system and method for capillary electrophoresis are provided foranalyzing a macromolecule prepared from a complex liquid mixture. Inparticular applications, methods and apparatus are provided forseparating and analyzing a solution containing a denatured macromoleculeby employing a stationary capillary electrophoresis apparatus.

An apparatus for capillary electrophoresis includes an inlet chamber anda capillary electrophoresis column. One end of the column is fixed atthe interior of the inlet chamber. The column has a length of at leastabout 20 centimeters. Also included is a liquid source adapted forautomatic control. The liquid source supplies a liquid sample through aninput valve into the inlet chamber so that the sample is in fluidcommunication with the end of the column.

A method for capillary electrophoresis includes automatically supplyingthe liquid sample to the apparatus.

The methods and apparatus disclosed herein provide significantadvantages to analyzing a macromolecule prepared from a complex liquidmixture by capillary electrophoresis. Fixing the column to the inletchamber provides durability compared to robotic CE systems, and removesthe need for calibration of the robotic system when a column isreplaced. Furthermore, the fixed chamber coupled with valved inputallows the system to exclude the liquid sample from the exteriorenvironment and allows rapid sampling and high throughput compared tochip based CE systems. The longer CE column allows better separation andhigher electric fields compared to chip based systems. Furthermore, thecombination of high throughput with provision of automatic controlallows analysis at a higher rate, enabling improved analysis and controlof time-sensitive bioreactor processes.

The combination of the stationary CE system with automated macromoleculesample preparation leads to efficient, high-throughput analysis ofmacromolecules from complex bioreactor mixtures. Rapid, sequentialsampling and analysis can be conducted without contamination or manualintervention. A sample can be automatically filtered and delivered tothe CE column for analysis, without being exposed to the environment.The combination of cleaning solution reservoirs allows the system to beautomatically cleaned in preparation for the next sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 depicts an example of a process for macromolecule preparation100.

FIG. 2 depicts typical steps that can be included in a macromoleculesample preparation process 200 operating on a mixture 202.

FIG. 3 depicts a hypothetical example comparing two samplingfrequencies, wherein a lower sampling frequency versus time (squares)can miss details in the level of a desired macromolecule versus time(solid line) in a reaction mixture, compared to a higher samplingfrequency (circles).

FIG. 4 depicts a schematic of steps that can be included in preparing amacromolecule sample.

FIG. 5 depicts an apparatus 500 that can conduct the steps in FIG. 4.

FIG. 6 depicts the lysis module 508.

FIG. 7A depicts rough separation circuit 700, containing asepticseparation circuit 752.

FIG. 7B depicts an aseptic fluidic interface apparatus 752.

FIG. 7C depicts apparatus 752 with a relief valve 758, overflowreservoir 760, and filter 766.

FIG. 7D depicts apparatus 752 wherein inlet and outlet valves 703 and702 can be double isolated gate valves.

FIG. 8 depicts desalination/fine filtration circuit 800.

FIG. 9 depicts denaturation circuit 900.

FIG. 10 depicts the separation of a small molecule 1002 and a largemolecule 1004.

FIG. 11 depicts a schematic of an electrophoresis apparatus 1100.

FIG. 12 depicts steps that can be included in analysis by stationarycapillary electrophoresis.

FIG. 13 depicts a stationary capillary electrophoresis circuit 1300 thatcan be controlled to conduct the steps in FIG. 12.

FIG. 14 depicts a more detailed schematic of the capillaryelectrophoresis circuit.

FIG. 15 depicts a block diagram of a preferred apparatus 1500.

FIG. 16 is a block diagram of a system including the subsystemsdescribed above and a local user interface for providing operationalinput.

FIG. 17 is a network diagram including multiple systems of FIG. 16connected to remote computing devices across a network.

FIG. 18 is a block diagram of an industry model in which a business maydistribute the system of FIG. 16.

FIG. 19 is a generalized flow diagram of a business method used in theindustry model of FIG. 18.

FIG. 20 is a flow diagram of a process used by the manufacturer of thesystem in FIG. 18.

FIG. 21 is a flow diagram of a process used by a customer in FIG. 18.

FIG. 22 is a detailed flow diagram of process steps in the flow diagramof FIG. 21.

FIG. 23 is a flow diagram of a process also used by the customer in FIG.18.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The methods and apparatus disclosed herein are generally related toanalyzing a sample of a molecular analyte, e.g., a macromolecule, from acomplex liquid mixture. The invention has particular application toautomated methods and apparatus for capillary electrophoretic analysismacromolecules, e.g., proteins, from a complex bioreactor liquidmixture.

Automated Macromolecule Preparation

FIG. 4 depicts a schematic of steps that can be included in preparing amacromolecule sample. The liquid, typically aqueous, mixture 202contains the macromolecule 104, and can also contain fine components213, e.g., salts, molecules smaller than the macromolecule, and thelike; and rough components 207, e.g., cells, cell fragments, particulatecontaminants, molecules larger than the macromolecule, and the like.

Macromolecule 104 can be dissolved in the liquid mixture, or can bepartially contained in cells, as depicted. Optional lysis step 204 lysesat least a portion of the cells to release macromolecule 104. Lysing canbe conducted using any method of lysing cells well-know to the art, forexample, heating, sonic disruption, addition of lysing agents, e.g.,detergents, changes in ionic strength, e.g., by dilution with water orcombination with a lysis buffer, and the like.

A rough separation step 410 applies the liquid mixture to a rough filter412, and a pressure differential across filter 412 directs at least aportion of the liquid, macromolecule 104, and the fine components 213through the filter, separating at least a portion of rough components207 at rough filter 412. Rough filter 412 can be selected to remove atleast a portion of components that are larger than the macromolecule,e.g., greater in diameter or molecular weight. Preferably, rough filter412 removes components that are greater in molecular weight than themolecular weight of the macromolecule by about 150%, more preferablyabout 125%, even more preferably about 110%, and most preferably, about105%. In other aspects, rough filter 412 can be selected to remove atleast a portion of components that are greater in diameter than about 60μm, more preferably about 30 μm, even more preferably about 10 μm, ormost preferably about 5 μm.

A fine separation step 414 applies the liquid mixture to a fine filter416, and a pressure differential across the filter directs at least aportion of the liquid and the fine components 213 through the filter towaste 418, separating at least a portion of macromolecule 104 at thefilter. Fine filter 416 can be preferably selected to remove at least aportion of components that are smaller than the macromolecule, e.g.,salt components. Preferably, the fine filter removes components thathave a molecular weight that is a fraction of the molecular weight ofmacromolecule 104 of preferably about 50%, more preferably about 75%,even more preferably about 90%, and most preferably, about 95%.

One skilled in the art will recognize that the separation steps can beconducted in any order, for example, fine separation 414 can beconducted before rough separation 410. Preferably, the steps areconducted in the order depicted in FIG. 4.

The liquid mixture remaining at the filter now has a greaterconcentration of macromolecule 104, and a reduced concentration ofsoluble fine components 213, e.g., salts. In step 420, the liquidmixture can optionally be combined with additional buffer 422 to adjustthe concentration of macromolecule 104 and other components, e.g., ions.Buffer 422 can contain pH buffer, other ionic buffers, filtration aids,denaturation agents, organic solvents, pure water, and the like.

One skilled in the art will recognize that in step 420, buffer 422 canbe added to either side of filter 416. Preferably, buffer 422 can bedirected through fine filter 416 by applying pressure differentialacross filter 416. This can dislodge portions of macromolecule 104 thatcan become attached to fine filter 416 in fine filtration step 414.Also, one skilled in the art will appreciate that steps 414 and 420 canbe repeated, providing greater separation of macromolecule 104 from finecomponents 213.

The concentration of the salt components is preferably reduced in steps414 and/or 420 by at least 50%, or more preferably, by at least 75%, ormost preferably, by at least 90%.

The concentration of the macromolecule is preferably increased by steps410 and/or 414 by at least 50%, or more preferably, by at least 100%, ormost preferably, by at least 200%.

Optional denaturation step 218 accepts the liquid mixture and at leastpartially denatures macromolecule 104 to prepared macromolecule 104′.The denaturation step can employ denaturing agent 220 and/or a heatingstep. The denaturing step 218 heats the macromolecule with denaturationagent 220 to, for example, from about 70° C. to about 100° C. for about60 to about 600 seconds; more preferably, from about 80° C. to about100° C. for about 120 to about 450 seconds; or even more preferably,from about 85° C. to about 95° C. for about 250 to about 350 seconds.Preferably, denaturation step 218 heats the macromolecule and thedenaturation agent to about 90° C. for about 300 seconds.

FIG. 5 depicts an apparatus 500 that can conduct the steps in FIG. 4.Sampling valve 502 opens to reactor site 102 through rough filter 412.Pump 506 draws the sample through optional lysis unit 508, where lysisstep 204 can be performed, and then through rough filter 412, removingrough components from the liquid mixture, i.e., step 410. Valve 502closes to filter 412 and valves 510 and 516 open, allowing pump 506 toreverse direction and drive the liquid mixture against fine filter 416,passing fine components through fine filter 416 and valve 516 to waste514, i.e., step 414.

Valve 516 can be closed and pumps 506 and 518 can be operatedcooperatively, i.e., pump 506 pushing and pump 518 pulling, to direct aportion of the liquid containing fine components 213 through fine filter416. When a portion of the liquid mixture has traversed filter 416,valve 510 closes and valve 516 opens, and pump 518 reverses to directthat portion to waste site 514, after which valve 516 closes. Asalternatives, only one of pumps 506 and 518 can be employed to direct aportion of the liquid containing fine components 213 through fine filter416.

To perform step 420, valve 520 can open and pump 506 can direct theremaining liquid mixture containing macromolecule 104 to denaturationvessel 526. Preferably, however, valve 522 opens and pump 518 draws aportion of buffer from reservoir 524. Valve 522 closes, valve 510 opens,and pump 518 directs the buffer through filter 416. Preferably, pumps518 and 506 operate cooperatively to direct the buffer through filter416, and pump 506 then directs the mixture through valve 520. Additionof the buffer through the filter can dislodge portions of macromolecule104 that may become associated with fine filter 416 in step 414.

Next, pump 506 drives the combination of macromolecule 104 to optionaldenaturation vessel 526, i.e., performing step 218, whereupon thedenatured macromolecule 104′ can be then directed to analysis site 106.

One skilled in the art will recognize that variations are possible inapparatus 500. For example, one or more of the valves, depicted astwo-way valves, could be combined into a single multifunction valve. Theplacement of various elements can be varied; for example, valve 502 canbe placed before rough filter 412, and the like.

FIG. 6 depicts lysis module 508. Pump 600 operates to draw liquidmixture 202, including cells, from reactor 102 through valve 602. Valve602 closes, valve 604 opens, and pump 600 draws a lysis buffer fromreservoir 606, lysing at least a portion of the cells in mixture 202.Pump 600 then directs lysed mixture 202′ through valve 608 to secondstage rough filter 412, preferably through a first stage rough filter610.

Aseptic Fluidic Interface coupled to Macromolecule Preparation Apparatus

FIGS. 7A-C, 8, and 9 depict a more detailed schematic of one embodimentof the invention. A controller 701 is coupled to the various pumps,valves, sensors, heating and cooling elements to provide automaticcontrol of the system. The controller may, for example, be a specialpurpose microprocessor based system or a general purpose computer.

FIG. 7A depicts rough separation circuit 700. The liquid mixture can bedrawn from bioreactor sample site 102 by opening valves 702, 703, and502 while closing valves 704, 706, 708, 710, 712, and 714 along thefluid stream. The line from site 102 includes a liquid/air trap region715, including waste valve 704, waste site 720, and flow sensor 718.

By operating rough pump 506, which is preferably a syringe pump, asample of liquid mixture 202 can be drawn from reactor site 102 at aflow rate of about 2 mL/min to reach a volume of about 10 mL. Thisaction draws the liquid mixture through initial filtering stepsinvolving first and second stage rough filters 610 and 412. Preferably,first stage rough filter 610 can be selected to remove rough components60 μm or larger, and second stage rough filter 412 can be selected toremove rough components 5 μm or larger. By closing valve 502, therough-filtered liquid mixture can be isolated in the syringe chamber ofrough pump 506.

Rough separation circuit 700 includes a number of valved reservoirs tosupply various standards, buffers and cleaning agents, including sizestandard reservoir/valve 728/706, cleaning solution reservoir/valve730/731, run buffer reservoir/valve 732/733, isopropyl alcoholreservoir/valve 734/735, and clean water reservoir/valve 736/737,wherein the amount of buffer drawn can be measured at flow sensor 738.One skilled in the art will recognize that a range of useful solventsand buffers can be employed for cleaning, for standardization, forstorage, for aiding filtration, and the like. For example, a sizestandard buffer can be used in a calibration run of the apparatus todetermine the separation performance of the apparatus. The size standardbuffer can contain a range of components of known size, at knownconcentrations, i.e., where size can include weight, molecular weight ordiameter of the components. The apparatus can be controlled toself-clean by employing a cleaning solution, preferably cleaning theapparatus between each run of a sample of the liquid mixture. Organicsolvents, e.g., isopropyl alcohol, can be employed as cleaning aids orto fill the fluid-handling elements of the apparatus when the apparatusis inactive for an extended period. Clean water and run buffer can beused to dilute the liquid mixture, to adjust the concentration of ions,to aid fluid flow, and the like.

Rough separation circuit 700 also includes a number of pressuretransducers 740, 742, 744 and 746, whereby the pressure in therespective portions of the circuit can be measured; compressed air orsteam 722,723,724, and 748 that can be employed for cleaning or purgingthe system; flow sensor 750; and waste site 792. The downstream boundaryof rough separation circuit 700 is valve 714, through which the liquidmixture can be directed to the desalination/fine filtration circuit 800.

Aseptic Fluidic Interface Apparatus

FIG. 7B depicts in detail an aseptic fluidic interface apparatus 752that can be used to provide a fluidic interface between two bioprocesssystems, a biofluid source site and a biofluid process site, e.g.,bioreactor 102 and apparatus 753 (the balance of rough separationcircuit 700).

Inlet valve 703 is coupled to a biofluid source site, e.g., bioreactor102. A sampling conduit 754 extends from inlet valve 703 to outlet valve702. Outlet valve 702 is coupled to a biofluid process site, e.g., theapparatus 753. Trap 715 is located at sampling conduit 754. Waste valve704 is located at a waste conduit 756, and extends from conduit 754 towaste site 720. A wash fluid source is coupled to at least one of theinlet and outlet valves 703 or 702, e.g., as depicted, reservoir/valve728/706 is coupled through outlet valve 702. The valves are all adaptedfor automatic control.

Trap 715 is a portion of conduit 754 that is lower in height than eitherend of conduit 754, e.g., so that fluid in conduit 754 tends to collectthere under gravity. The lowest portion of the trap is generally belowthe lowest end of conduit 754 by a multiple of the conduit internaldiameter (or average internal diameter) of at least about 3 times, moretypically, at least about 5 times, even more typically about 10 timesand preferably at least about 20 times. The trap is typically a U-shapedportion of conduit, and the ends, e.g., at input and output valves 703and 702, are preferably at the same height. Waste valve 704 can becoupled to any point in the trap but is typically coupled to the lowestpoint of the trap. The volume of the conduits bounded by valves 703,702, and 704, e.g, the volume of the trap, in milliliters, is related tothe cross-sectional area of the conduit by a multiplier that istypically less than about 15, more typically less than about 10, evenmore typically less than about 5, still more typically less than about2, and preferably less than about 0.5. For example, for a conduit with across sectional area of 1 millimeter², if the factor is 10, the volumeis less than about 10 milliliters; if the factor is 2, the volume isless than about 2 milliliters; and the like.

Aseptic fluidic interface apparatus 752 can control fluid transferbetween the two systems so that fluid is transferred in a particulardirection at particular times, e.g., only from bioreactor 102 toapparatus 753 during sample collection. For example, automatedcontroller 701 can communicate electronically with the valves,collecting fluid sample from bioreactor 102 by opening inlet valve 703,directing the sample to apparatus 752 by opening outlet valve 702 whilewaste valve 704 is closed. Reactor 102 and apparatus 753 can be isolatedby closing inlet valve 703 and outlet valve 702, and trap 715 andsampling conduit 754 can be drained to waste site 720 by opening wastevalve 704. Before transferring a sample, preferably as part of eachsample cycle, sampling conduit 754 can be cleaned by opening waste valve704 and directing a wash fluid through at least one of inlet and outletvalves 703 or 702 and subsequently through waste valve 704 to waste site720, e.g., from wash reservoir 728 through outlet valve 702. An optionalflow sensor 718 can be located in apparatus 752, typically at samplingconduit 754 or waste conduit 756, preferably at waste conduit 756between trap 715 and waste site 720. Flow sensor 718 can be employed bycontroller 701 to sense for fluid flow, particularly when the twobiofluid sites, e.g., bioreactor 102 and apparatus 753, are isolated. Ifflow is sensed during isolation, a possible backflow condition can beindicated. As used herein, “backflow” means undesirable fluid flow inthe system, e.g., due to failure of valves 703 or 702 to close, and thelike. Backflow can lead to cross-contamination, loss of valuablebioreactor fluid, and the like.

Apparatus 752 preferably controls fluid transfer so the transfer isaseptic. As used herein, aseptic means that the integrity of the sampleis maintained. For example, the sample can contain microorganisms,macromolecules, fluids, salts, etc., e.g., those present in bioreactor102. However, external contaminants, e.g., microorganisms,macromolecules, and other chemical, biological or particulatecontaminants from the external environment can be excluded from theapparatus. Furthermore, in the wash process, the residue from eachprevious sample can be removed from the apparatus. For example, when aprocess is sampled over time to determine the concentration versus timeof a macromolecule, it can be desirable to remove traces of themacromolecule from a previous sample so that the accuracy of a futuresample is not affected. Similarly, microorganisms can be removed toavoid a microorganism lodging in the apparatus and excreting amounts ofthe macromolecule which could affect accurate measurement.

FIG. 7C depicts an aseptic fluidic interface apparatus 752 with a reliefvalve 758, overflow reservoir 760, and filter 766, all located on reliefconduit 764. Flow sensor 718 can optionally be located on relief conduit764 as shown. Relief conduit 764 extends from waste conduit 756 at apoint between trap 715 and waste valve 704, and ends in fluidcommunication with the external environment through filter 766. Filter766 excludes at least a portion of external contaminants from at least aportion of the relief conduit. The filter can be located anywherebetween valve 758 and the distal end of conduit 764, preferably at theend as depicted in FIG. 7C. Typically, the filter is selected to excludemicroorganisms and particulate contaminants, e.g., the filter excludescontaminants having a diameter greater than about 1 μm, more typicallygreater than about 0.5 μm, and preferably greater than about 0.2 μm.Overflow reservoir 760 can be located anywhere between valve 758 and thedistal end of conduit 764, preferably between filter 766 and valve 758as depicted in FIG. 7C. Flow sensor 718, which can be located anywherein apparatus 752, is typically at waste conduit 756 or relief conduit764. If the overflow elements are employed, flow sensor 718 is typicallyat conduit 764 as shown, preferably between valve 758 and reservoir 760.A second filter 768 can be employed at conduit 764, e.g., between valve758 and trap 715. Filter 768 is sized smaller than filter 766, i.e.,excludes at least a portion of contaminants that pass through filter766. For example, filter 768 is typically sized to exclude particlesless than about 75% of the size excluded by filter 766, more typically,less than about 50% of the size excluded by filter 766, an preferably,less than about 25% of the size excluded by filter 766.

Automated controller 701 directs wash fluid into the sampling conduitthrough at least one of inlet and outlet valves 703 or 702, preferablyoutlet valve 702. A wash fluid can be one or more fluids, e.g. a gas, avapor, a liquid, a supercritical fluid, a combination, and the like. Forexample, gases can include compressed air, oxygen, nitrogen, noble gasesnitrous oxide, ethylene oxide, carbon dioxide, and the like; vapor caninclude steam or vaporized solvents; liquids can include water, aqueoussolutions of buffers, antiseptics, detergents, and the like; solvents,e.g., organic solvents such as alcohols, ethers, ketones, polar aproticsolvents, and the like; and supercritical fluids can include carbondioxide, water, and the like. Typically, the wash fluid is sterile. Morethan one fluid can be employed, for example, the apparatus can beflushed with an aqueous cleaning solution, steam, and then drycompressed air. Preferably, at least one wash fluid is antiseptic orsterilizing, i.e., is able to kill microorganisms.

The automated controller can direct the wash fluid along a number ofpaths. Starting from reservoir 728, the fluid can be directed throughoutlet valve 702, From there, it can be directed through valve 704 towaste 720, or through valve 758 to overflow reservoir 760, or throughinlet valve 703 back into reservoir 102.

Automated controller 701 is typically employed with the wash fluid toreduce bacterial count, macromolecule contamination, and/or othercontamination to acceptable levels. An “acceptable level” ofcontamination is that level of contaminants that do not have ameasurable adverse effect on the bioprocess site. For example,macromolecule contamination is typically reduced below the detectionlevel of an analysis circuit coupled to the system. Contamination of anyportion of the system can be measured using rinse water, e.g., byfilling that portion with rinse water, letting stand at 20° C. for 1minute, and then analyzing the rinse water for the concentration ofmacromolecules or bacteria. Typically, washing can reduce bacterialcontamination, e.g., the number of bacterial colony forming units permilliliter of rinse water to less than about 100, more typically, toless than about 50, and preferably, to less than about 10. Generally,washing can reduce macromolecule contamination in rinse water to lessthan about 10 parts per million (ppm), more typically, to less thanabout 1 ppm, even more typically, less than about 0.1 ppm, andpreferably, to less than about 0.01 ppm.

FIG. 7D depicts still other options for apparatus 752. One or morevalves, e.g., inlet and outlet valves 703 and 702 can be double isolatedgate valves. As used herein, a double isolated gate valve is a singlevalve unit that can be considered as two coupled three-way valves.Typically, a double isolated gate valve has minimal dead volume betweeneach of its three-way valves. These valves can allow other options forfluid flow. For example, wash fluid can be directed into the systemthrough one such valve, e.g., into outlet valve 702. The wash fluid canthen be directed out of the remaining output of double isolated outletgate valve 702 to waste site 720, or alternatively, into samplingconduit 754, up to double isolated inlet gate valve 703, and then towaste site 720.

FIG. 8 depicts desalination/fine filtration circuit 800, which followsrough separation circuit 700 from FIG. 7A. The liquid mixture, nowseparated from at least a portion of rough components, can be acceptedfrom rough separation circuit 700. Valves 714 and 510 are opened andbranch valves 710, 712, 802, 804, 516, and 520 are closed. Rough pump506 can be emptied at about 1.5 mL/min to a total of about 7.5 mL. Atthe same time, fine pump 518 can be controlled to draw the plunger backat about 1.5 mL/min. This creates the force to direct the liquid mixtureout of rough pump 506, across fine filter 416 and into the syringechamber of fine pump 518. Fine filter 416 and rough pump 506 retain themacromolecule while passing a solution of fine, e.g., salt componentsthrough and into the syringe chamber of fine pump 518.

Next, valves 510 and 714 are closed, and valve 516 can be opened. Finepump 518 can be activated to push the syringe plunger contents (about7.5 mL) at about 1.5 mL/min and direct at least a portion of the liquidmixture containing fine components, e.g., sodium chloride, to waste site514. Flow sensor 515 can be employed to monitor the liquid sent towaste.

Next, a desalination buffer can be loaded by opening sample buffer feedvalve 522, and manifold feed valve 806 and drawing sample buffer fromreservoir 524 into fine pump 518. About 7.5 mL of sample buffer can bedrawn from reservoir 524, after which sample buffer feed valve 522 andmanifold valve 806 are closed. The amount of buffer drawn can bemeasured at flow sensor 808. Other buffers can be provided, for example,valved reservoir 810/811 can provide, e.g., a pH buffer, pure water,etc.

As described in FIG. 5, to perform step 420, valve 520 can open and pump506 can direct the remaining liquid mixture containing macromolecule 104to denaturation vessel 526.

Next, valves 522 and 806 close, valves 510 and 520 open, and pumps 518and 506 work together to direct the mixture to denaturation circuit 900,where valves 902, 904, 906, and 908 are closed (see FIG. 9). Pumps 518and 506 operate at a rate of about 1.5 mL/min. Typically, rough pump 506will push a total of about 2.5 mL, while fine pump 518 will push a totalof about 7.5 mL.

Fine separation circuit 800 also includes a number of pressuretransducers 812, 814, and 816, whereby the pressure in the respectiveportions of the circuit can be measured. Valve 802 can providecompressed air or steam for cleaning or purging the system.

FIG. 9 depicts denaturation circuit 900. Denaturation vessel 526 ispreferably a 10 mL stainless steel vessel that contains both heating andcooling coils. The mixture can be heated until at least partialdenaturation occurs, for example, heating to at least about 70° C. forabout 90 seconds, or more preferably, heating to about 90° C. for about300 seconds. Subsequently, the cooling coil can be operated to cool thesample to about 25° C.

Upon denaturation, denatured macromolecule 104′ can be removed byactivating denaturation pump 916, opening valve 902 and opening eithervalve 912 or 913 to allow the liquid mixture to be drawn from denaturingvessel 526. The concentration of ions in the mixture, for example, theconcentration of hydrogen ions, i.e., the pH of the mixture can bemonitored at sensor 909. Subsequently, the mixture containing denaturedmacromolecule 104′ can be passed through a precipitate filter 932 byclosing valves 918, 920, 922, 926, and 928, and opening valves 908 and924. Pumps 916 and 930 operate cooperatively, i.e., pump 916 pushing andpump 930 pulling, to drive the liquid mixture against precipitate filter932. Precipitate filter 932 can be selected to exclude insolublecomponents that can precipitate during the denaturation step.Preferably, filter 932 excludes insoluble components greater in diameterthan about 1 μm, more preferably about 0.6 μm, and most preferably about0.45 μm. Once the mixture is filtered of at least a portion ofprecipitate and is in the syringe chamber of pump 930, valve 924 can beclosed and analysis site feed valve 928 can be opened, and pump 930 candirect the mixture containing prepared macromolecule 104′ to analysissite 106.

Denaturation circuit 900 also includes denaturation vessel valve 934;compressed air or steam inlet valves 904, 920, and 926; flow sensors 936and 938; waste sites 940 and 942; and pressure transducers 944 and 946.

Stationary Capillary Electrophoresis

FIG. 12 depicts steps that can be included in analysis by stationarycapillary electrophoresis. A liquid sample 1202 includes one or moremolecular analytes 1204 and other components 1206. As used herein, amolecular analyte is any molecule that is soluble or suspended in theliquid sample and has an electrophoretic mobility that is different fromother components 1206 in the liquid sample. The molecular analyte can beany molecule, e.g., inorganics, small molecule organics, biomolecules,synthetic polymers, biopolymers, proteins, peptides, amino acids,nucleic acids, and the like. Preferably, the molecular analyte is amacromolecule, i.e., macromolecule 104, and most preferably, separatedmacromolecule 104′.

The liquid sample is introduced to the end of an electrophoresis column1106 by pressure or electro-kinetic injection in step 1208. A buffer1210 that contains, for example, electrolytes know to the art to besuitable for capillary electrophoresis can be added. Additionalcomponents of the buffer known to the art can include organic solvents,e.g., acetonitrile; additives which can act to reduce electroosmoticflow; electrophoretic flow modifiers, i.e., ionic agents that complexwith molecular analytes to change electrophoretic mobility;spectroscopic or radioactive tags; and the like.

A voltage differential is applied across the column in step 1212,causing the molecular analyte to separate from other components. Inoptional step 1214, pressure differential can be applied to the columnto cause liquid in the column to flow. For example, the effective lengthof the column can be increased, e.g., by conducting a partialelectrophoretic separation step 1212, pausing, performing optional step1214 to flow liquid in the column in a direction contrary to theelectrophoretic flow, and then resuming electrophoretic flow step 1212.One skilled in the art will appreciate that steps 1212/1214 can berepeated numerous times. Once the molecular analyte is separated, it canbe analyzed in detection step 1216, either while still in the column bydetector 1110, as depicted, or after extraction from the column.

FIG. 13 depicts a stationary capillary electrophoresis circuit 1300 thatcan be controlled to conduct the steps in FIG. 12. The inlet chamber1102 is supplied with the liquid sample by pump 1302 through inlet valve1304 from liquid sample source 1301. Optional precipitate filter 1303can be employed to separate insoluble precipitates from the liquidsample by employing pump 1302 to apply the liquid sample to filter 1303with a pressure differential across the filter.

Each chamber can be supplied independently by a buffer reservoir 1306through valves 1308 and 1310. Each chamber can be independently drainedvia valves 1312 and 1304 to waste sites 1316 and 1318. Pump 1320 candraw filtered air through air inlet valve 1326 and air source 1328, andindependently direct the air to chambers 1102 and 1104 through valves1322 and 1324. The valves, pumps, optional electrophoresis power supply1108, and optional detector 1110 are adapted to be controlled by anoptional controller 1330.

The capillary electrophoresis column 1106 is coupled with the interiorof each chamber so that liquid in each chamber can be placed in fluidcommunication with the respective end of column 1106. Preferably, thecolumn has a length of at least about 20 centimeters, more preferably atleast about 30 centimeters, and most preferably, at least about 50centimeters.

The optional detector 1110 can be any detection method known to the artfor detection of molecular analytes, for example,absorbance/transmission of radiation, e.g. ultraviolet/visible light;fluorescence detection; refractive index detection; electrochemicaldetection; mass spectrometric detection; detection of electron ornuclear magnetic resonance; flame ionization detection; binding, e.g.,in an enzyme or antibody assay; detection of a spectroscopic orradioactive label; and the like. When optional detector 1110 is anoptical detector, it can be configured to detect molecular analytes thatare inside column 1106. Or, fractions can be collected from themolecular analytes exiting column 1106, e.g., at outlet chamber 1104,and the fractions can be analyzed separately from the column.

Additionally, each chamber can be barometrically sealed, i.e., they canbe pressurized or depressurized. For example, valves 1304, 1308, 1312,1324, and 1326 can be closed, valve 1322 can be opened, and pump 1320can pressurize inlet chamber 1102. If the pressure in chamber 1102 isgreater than the pressure in outlet chamber 1104, a high to low pressuredifferential results across the length of capillary electrophoresiscolumn 1106. Alternatively, pump 1320 can reduce the pressure in chamber1102 to less than the pressure in chamber 1104, resulting in a low tohigh pressure differential, which can direct liquid from chamber 1102through column 1106 to chamber 1104. Or, the valves can be configured sothat pump 1320 can pressurize or depressurize chamber 1104. Optionally,separate independent pumps can be coupled with each chamber and thepumps can operate cooperatively, one pulling and the other pushing, tocreate a pressure differential across column 1106. Creation of apressure differential between chamber 1102 to chamber 1104 throughcolumn 1106 can be employed to fill, purge, or clean the column, or tomove fluid through the column, e.g., perform step 1214.

FIG. 14 depicts a more detailed schematic of the capillaryelectrophoresis circuit. The line between inlet valve 1304 and inletchamber 1102 can be supplied with compressed air by filtered air supply1402 through valve 1404. An additional optional air source 1406 andvalve 1408 is provided that can be employed to purge inlet chamber 1102and/or the waste line between valve 1312 and waste 1316. The waste fromboth chambers is provided with flow sensors 1410 and 1412, respectively.Pressure transducers 1414 and 1416 are provided to sense the pressure inthe apparatus. Along with reservoir 1306 are provided a valve 1418 andadditional valved reservoirs 1420/1421, 1422/1423, and 1424/1425. Thesereservoirs can supply water, buffer, cleaning solution, solvents,electrolytes, and the like, the flow of which can be sensed at flowsensor 1426. Additional buffer can be supplied to the outlet chamber1104 by reservoir 1428 through flow sensor 1430 and valve 1432.

Additionally, the level of fluid in chambers 1102 and 1104 can be sensedindependently by level sensors 1434 and 1436, respectively. Heatgenerated in column 1106 by the electrophoresis current can be removedby a heat exchanger 1438, which can be, for example, a cooling element,a thermoelectric element, and the like. Also, optional degas unit 1440can be employed to remove at least a portion of dissolved gases.

Automated System for On-Line Aseptic Sampling of Bioreactor Fluids,Macromolecule Separation, Denaturation, and Capillary ElectrophoreticAnalysis

FIG. 15 depicts a block diagram of a preferred apparatus 1500, whichcouples rough filtration circuit 700, desalination/fine filtrationcircuit 800, denaturation circuit 900, and capillary electrophoresiscircuit 1300 into an integrated system. That is, rough filtrationcircuit 700 inputs a complex liquid mixture 202 comprising amacromolecule, and separates the macromolecule from rough components.The mixture is directed to desalination/fine filtration circuit 800, andthe macromolecule is separated from at least a portion of finecomponents, including salt components. The mixture is then directed todenaturation circuit 900, where the macromolecule is denatured andseparated from any insoluble precipitates that form during denaturation.This creates a liquid sample, containing denatured macromolecule 104′,that can be directed to capillary electrophoresis circuit 1300. In thisview, several elements described in preceding Figs. can be synonymous,e.g., pumps 930 and 1302 can be the same pump; valves 928 and 1304 canbe the same valve; filters 932 and 1303 can be the same filter; andautomated controllers 701 and 1330 can be the same controller.

One skilled in the art will appreciate that the various elements ofapparatus 1500 can be integrated in different combinations. For example,each of the various individual elements can be integrated with abioreactor, for example, rough filtration circuit 700 can be integratedwith a bioreactor, or denaturation circuit 900 can be integrated with abioreactor, and the like. Combinations of the various elements van alsobe employed, for example, for a particular biofluid source that does notrequire rough filtration or denaturation, aseptic interface 752 can becombined with fine filtration circuit 800 and capillary electrophoresiscircuit 1300. In another example, a system that does not require finefiltration could employ rough separation circuit 700, denaturationcircuit 900, and capillary electrophoresis circuit 1300. In otherapplications, each circuit or apparatus can be used alone, or in otherlogical combinations. One skilled in the art will know which circuit orapparatus will be useful in any particular application.

In various embodiments, each of the individual elements in apparatus1500 and various combinations thereof can be coupled “on-line” to anoperating bioreactor. As used herein, “on-line” means that the apparatuscan draw samples directly from the reactor into the apparatus, i.e., thesample is drawn directly into the apparatus without exposure to theexternal environment and without involving a transfer using a discretesample container, e.g., a sample vial.

Another feature of particular embodiments of combinations of two or moreof the elements of apparatus 1500 is that each combination can becoupled to an operating bioreactor to form an integrated system. Thismeans that the sample is in complete custody of the system, i.e., iscontrolled to be free from exposure to the external environment, fromthe bioreactor to the final operation on the sample (e.g., analysis ofthe prepared macromolecule at capillary electrophoresis circuit 1300).Furthermore, “integrated” can mean that the various circuits andapparatuses are controlled by the automated controller to operate in acoordinated fashion.

Still another feature of various embodiments of the elements ofapparatus 1500 and their combinations is that each can be coupled to anoperating bioreactor to handle “raw” fluids, i.e., complex liquidmixtures containing one or more components typically found in abioreactor, for example, cells, cellular debris, cell organs, cellfragments, salts, macromolecules including proteins, DNA, RNA, and thelike. A “raw” fluid is taken directly from a reactor, typically anoperating reactor, without any preprocessing.

In particular embodiments, the capillary electrophoresis circuit 1300can be controlled to partially or completely exchange the fluid insidethe capillary electrophoresis column in place, i.e., the column canremain fixed with respect to one, or preferably both of the inlet andoutlet reservoirs.

In various embodiments, the inside diameter of the capillaryelectrophoresis column is at least about 50 μm, more typically at leastabout 75 μm, and even more typically at least about 100 μm. One skilledin the art will know that values larger than 1 mm for the insidediameter of the capillary are possible, but can face diminishing returnsin terms of efficiency. In a particular embodiment, the inside diameterof the capillary is from about 50 μm to about 150 μm, or moreparticularly, from about 100 μm to about 125 μm.

In various embodiments, each system, circuit and apparatus can drawsample volumes from at least about 0.1 mL to at least about 25 mL, andmore typically between about 0.5 mL and about 10 mL. In a particularembodiment, the sample volume is between about 0.75 and about 5 mL.

The inside diameter of the conduits employed in the various circuits inthe system, excluding the capillary itself, can be in various ranges.The inside diameters can be different in different portions of thesystem. The inside diameters are typically in a range of from about 0.5to about 10 millimeters (mm), more typically between about 0.75 andabout 5 mm, even more typically between about 0.75 and about 2 mm, andpreferably between about 1 and about 2 mm.

The “pressure differential” employed to direct components at or througha filter can be estimated by one skilled in the art by consideringrelevant system characteristics such as filter pore size, fluidviscosity, approximate concentration of material larger than the filterpore size, time to filter a particular volume, flow rate, and the like.One skilled in the art will know how to use such characteristics tochoose an appropriate pressure differential based on the desired filterperformance and flow rate. Typically, the pressure differential acrossthe filter is between about 500 and about 7000 millibar, more typicallybetween about 1000 and about 5000 millibar, or even more typicallybetween about 1500 and 3000 millibar. A “pressure differential” can becaused by pressurizing one side of the filter, depressurizing on oneside of a filter, or a combination of pressurizing one side anddepressurizing the other side in a “push-pull” fashion.

As used herein, the filters are employed as “direct flow” or “dead-end”filters, and filtration methods employed herein are “direct flow” or“dead-end” filtration methods. This means that during filtration, thepressure differential applied causes the liquid mixture being filteredto be applied directly to the filter, i.e., in a direction substantiallyperpendicular to the face of the filter.

Another particular embodiment of the filters and filtration methodsemployed is a “back-flushing” capability. That is, each filter can becleaned by directing a fluid, e.g., a buffer, a cleaning fluid, water, asolvent, a desalination buffer, a denaturation buffer, combinationsthereof, and the like through the filter in a direction opposite to aprevious filtration step. For example, a filter which becomes cloggedwith debris after a filtration step can be cleaned, at least in part, bydirecting a fluid through the filter in a direction opposite to thedirection of the preceding filtration step.

The controllers 701/1330 may receive operational input 1615 from anexternal source, such as a local user interface (not shown). Thecontroller(s) 701/1330 process the operational input 1615 to sendcommands or queries 1505 to the circuits 700, 800, 900, or 1300 and, insome embodiments, receive responses 1510 from these circuits.

FIG. 16 is a block diagram of an overall system 1600 shown in thecontext of additional external systems and input/output data related tothe system 1500.

From a macromolecule processing point of view, this overall system 1600refers to the system 1500, the liquid mixture 202 that includes amacromolecule of interest, and the prepared macromolecules 104′.

From a controls point of view, the overall system 1600 includes thesystem 1500, controller(s) 701/1330 in the system 1500, and local userinterface 1605 connected to the system 1500 via a bus or local areanetwork 1607.

The controllers 701/1330 may include executable instructions, providedin the form of software or firmware, which is preferably unchangeable bythe user of the system 1500. Such unchangeable software may be referredto, and is referred to hereafter, as “compiled” software, meaning thatsource code was compiled (e.g., compiled C code), and the compiledsoftware exists only in a form usable by the controllers 701/1330.Source code may be provided to the user for re-compiling to facilitatemodification of the configuration or general operation of the system.However, re-compiling may cause a re-validation and/or re-approval ofthe system 1500 to be required before further usage, which is discussedlater in reference to FIGS. 18-23.

The operational input 1615 can be provided or written by a designer orend user of the system 1500 without having to recompile the compiledsoftware. The operational input 1615 typically provides specificoperational instructions to customize operation of the system, which maybe limited by the compiled software according to a predefined set oflimits.

The local user interface 1605 may include a general purpose computer orcustom-designed computer specific for operating the system 1500. Thelocal user interface 1605 may send the operational input 1615 to thecontrollers 701/1330, which process the operational input 1615 using thecompiled software 1610. Responsively, the compiled software 1600 maysend commands or queries 1505 to the system components 700, 800, 900,and 1300 via an internal bus or network (not shown).

The compiled software 1610 may be stored locally or downloaded acrossthe network 1607 and is executed by the controllers 701/1330. Thecompiled software 1610 may also be permanently stored in the controllers701/1330 through the use of firmware, Field Programmable Gate Arrays(FPGA's), Read-Only Memory (ROM), and so forth.

The controllers 701/1330 may also collect data, such as the productiondata 1625 and/or electrophoresis data 1620, during operation of thesystem 1550. These data 1620, 1625 may be sent via the network 1607 tothe local user interface 1605 for further processing or display to theuser via a Graphical User Interface (GUI), other display, such as LEDindicators, or output as sound, such as produced by an audiosynthesizer.

The electrophoresis data 1620 may include information regarding theprepared macromolecules 104′. For example, the electrophoresis data 1620may include the molecular weight of the sample and time corresponding tohow long the sample takes to travel across the length of theelectrophoresis column 1106.

The production data 1625 may include information regarding the system1500, such as calibration information, such as throughput/recovery andmolecular weight calibrations, equipment specifications, such ascapillary diameter, voltage levels, capillary length, cleaningsolutions, and number of usages since the last replacement of theelectrophoresis column.

The production data 1625 may also include information related to theproduction of the macromolecules from the liquid mixture 202, such asdiscussed in reference to FIG. 5.

The compiled software 1610 may have knowledge of a mapping, inaccordance with a known industry standard between the operational input1615 and system components 700, 800, 900, or 1300, includingsubcomponents, such as pumps, valves, heating or cooling elements,pressure sensors, etc. The compiled software 1610 is preferably testedand integrated by the manufacturer of the system 1500 in a manner alsoconsistent with known industry standards, such as American NationalStandards Institute (ANSI) (e.g., ANSI ‘C’ programming language).

For example, the manufacturer may validate the system by (i) inputting acomplete set of test vectors to the controllers 701/1330 in a testingphase of the system 1500 and (ii) observing activation/deactivation ofthe valves, pumps, etc. in accordance with the test vectors. Prior torelease of the compiled software with the system 1500, the compiledsoftware 1610 may be tested extensively for failure modes and/or errorchecking capabilities for detecting programmatic or out-of-range errorsidentified in the operational input 1615. Other forms of testing mayinclude providing test vectors with erroneous or harmful information toensure the compiled software 1610 handles these situations in a mannerthat protects the system 1500 or components therein.

In a preferred embodiment, when sold or released to a customer, thecompiled software 1610 is unchangeable by the customer. In other words,the customer cannot alter the compiled software 1610 and, therefore, thesystem 1500 continues to operate and be controlled in a manner testedand validated by the manufacturer of the system 1500. The operationalinput 1615, however, can be modified by the customer independent of thecompiled software 1610 to customize the operation of the system. Forexample, if a particular macromolecule requires additional filteringcycles or denaturation dwell time, the customer may customize theoperational input 1615 to provide such control.

The operational input 1615 may be declarative software instructions,where declarative software instructions are defined as instructions of arelational language or functional language, as opposed to an imperativelanguage, where imperative (or procedural) languages specify explicitsequences of steps to follow to produce a result. Declarative languages,in contrast, describe relationships between variables in terms offunctions or inference rules, and a language executor (i.e., interpreteror compiler) applies some fixed algorithm to these relations to producea result.

Thus, for example, the operational input 1615 may be softwareinstructions, such as BASIC software instructions, that are interpretedin a real-time or pseudo-real-time manner by the compiled software 1610.The operational input 1615 may also be forms of data streams that areproduced by a graphical user interface (GUI) and processed by thecompiled software 1610.

An example set of program instructions or portion of operational inputis listed below. The program instructions form a representative scriptfor operating an analyzer, such as the capillary electrophoresis circuit1300. The representative script may be referred to as a physical layerbetween the user and the compiled software 1610 to permit a chemist oroperator to program the operation in an english-like language thatprovides an intuitive understanding for the programming. Use of thistechnique permits the manufacturer of the system 1500 to “hard code” thephysical operation of the system 1500 while permitting the end user to“soft code” the operational input 1615 for customizing or modifying aprocess based on empirical or calculated process flows.

“%” Denotes comment and is not executed % script - flow to DesaltingFilter % % begin Push Sample to Desalt Filter Routine echo “Push Sampleto Desalt filter.” open_valve SV1022 % open ‘Pump- Desalt IsolationValve- SV1022’ (FIG. 8, valve 714) sleep 1.0 % Pause 1 second open_valveSV1026 % ‘open -- Pump 2 -Desalt Isolation valve- SV1020’ (FIG. 8, valve510) sleep 1.0 % Pause 1 second % watch_pressure_drop <pt1>, <pt2>,<time_in_seconds>, <warning_low>, <warning_high>, <error_low>,<error_high> watch_pressure_drop PT4, PT5, 10.0, 0, 10, −5, 20 % movesample across filter(s) by activating syringes start_syringe SY1, PUSH,7.5, 0.6 start_syringe SY2, PULL, 7.5, 0.6 wait_for_syringesend_pressure_drop PT4, PT5 % quit reading F3 pressure drop % Sample toDesalt Filter transfer complete - release valves close_valve SV1022 %close ‘Pump 1 - Desalt Isolation Valve- SV1022’ (FIG. 8, valve 714)sleep 1.0 % Pause 1 second close_valve SV1026 % close ‘Pump 2-DesaltIsolation Valve- SV1026’ (FIG. 8, valve 510) % end Push Sample to DesaltFilter Routine

The above script may be stored in the local user interface 1605 andprovided as the operational input 1615 to the controller 1330. Thecompiled software 1610 on the controller 1330 interprets the statementsin the above script to generate commands or queries 1505 to/fromcomponents in the capillary electrophoresis circuit 1300 or valves,syringes, etc. in a preceding circuit, such as the denaturation circuit900.

As described above, the compiled software 1610 includes softwareinstructions unchangeable by the user. The compiled software 1610interprets statements such as “open_valve SV1022” to mean “provide asignal to energize or deenergize the valve corresponding to the variableSV1022 in a manner such that the valve opens to allow a liquid source toflow into an inlet chamber.” Responsively, the compiled software 1610causes the controller(s) 701/1330 to produce signals that effect thisinstruction. Since the compiled software 1610 knows of thecorrespondence between the valve referred to as SV1022 in theoperational input 1615 to correspond with, for example, valve 714 (FIG.8), the user need only specify valve SV1022 to be sure that the correctvalve, valve 714, will be opened. Similarly, valve SV1026 corresponds tovalve 510 as shown in FIG. 8, so the “open valve SV1026” instructionwill be interpreted as such by the compiled software 1610, which, inturn, causes the controller(s) 701/1330 to generate an electrical signalthat energizes or de-energizes the valve 510 to produce the desired“open” state of the valve 510.

Continuing to refer to the script above, pressure sensors, whoseaddresses are known to the compiled software 1610 in connection with thevariable names PT4 and PT5, are addressed by the controller 1330executing the compiled software 1610 in response to receipt of theoperational input 1615 that includes the ‘watch_pressure_drop’statement. The addresses corresponding to the syringes SY1 and SY2 arealso known to the compiled software 1610 and addressed by thecontroller(s) 701/1330 to “push” (i.e., deliver volume) and “pull”(i.e., acquire volume) in response to receipt of the ‘start_syringe’statements listed above.

As should be understood from the above code, a “plain english” languageset of programming instructions may be supported for a user of thesystem 1500 for customizing the process for collecting electrophoresisdata 1620 from a sample of a processed macromolecule sample. Thevariable names (e.g., SV1022, SY1, PT4, etc.) may also be or includemnemonics or other forms of descriptors that are identified by thecompiled software 1610 and represent corresponding devices or subsystemsto be operated in a manner consistent with the command(s) associatedtherewith.

The correspondence information may be embedded directly in the code,stored as sets of constants or hard coded variables in the software, orstored in look-up table(s), list(s), such as arrays or linked lists, orcalculations used by the controllers 701/1330 to determine thecorrespondence between the variable names and elements correspondingthereto.

In this way, once testing of the compiled software 1610 has beencompleted, where the testing typically includes an exhaustive set oftest vectors that is consistent with a full range of possible inputsprovided by users of the system 1500, the manufacturer, customer, anduser of the system 1500 are assured that this correspondence is “fixed”such that inadvertent addressing errors by the controllers 701/1330 willnot be encountered, excluding electronics errors or failures. In otherwords, operational input 1615 that includes commands listed above in theexample script will result in a known and repeatable effect to ensureproper operation of the system 1500 for processing or analyzingmacromolecules.

FIG. 17 is a network diagram of a network 1700 that includes multiplemacromolecule systems 1500 connected to a central or distributed network1705. Each of the systems 1500 has a local user interface 1605, asdescribed above in reference to FIG. 16. In this embodiment, however,the systems 1500 or local user interfaces 1605 include interfaces (notshown) to receive operational input 1615 from a remote user interface1710 across the network 1705.

The remote user interface 1710 can be employed by a “central” operatorto control or monitor a distributed network of the macromolecule systems1500 for high yield production or analysis of macromolecules. Forexample, a large pharmaceutical company or manufacturer supplyingbiological product thereto may employ such a network for high volumeproduction.

Beyond the operational inputs 1615, the remote user interface 1710 mayalso request data from a remote processing/data stores device 1715,which is also coupled to the network 1705 for interfacing with thesystems 1500. The remote processing/data stores device 1715 may receivethe production data 1625 or electrophoresis data 1620 for processingthis data “off-line”. For example, the remote processing/data storesdevice 1795 may determine yields or quality of the macromoleculesprocessed by the systems 1500 and provide access to this data across thenetwork 1705, for example, to the remote user interface 1710 or any ofthe local user interfaces 1605. Thus, in response to the data request1720, the remote processing/data stores device 1715 may providerequested information 1725, including raw or processed data, across thenetwork 1705 in a typical data exchange manner, such as throughpacketized communications.

It should be understood that the network 1705 may include various formsof communication networks, such as a Public Switched Telephone Network(PSTN), wired or Wireless Local Area Networks (WLAN's), cellularnetworks, circuit switching networks, Voice-Over-Internet-Protocol(VoIP) networks, and so forth.

It should be understood that the compiled software 1610 operating in thecontrollers 701/1330 of the systems 1500 may be organized into multiplesoftware “units”, such as a system control unit, network interface unit,local interface unit, and so forth. In this way, the compiled software1610 can be updated with predetermined re-validation requirements tominimize future costs of maintaining the system 1500 by the customers.

Business Method

FIG. 18 is a schematic diagram of a business model 1800 in which amanufacturer of systems subject to approval by a regulatory body 1820operates. The regulatory body may (i) provide oversight of a system,such as the system 1600 discussed above, produced by the manufacturingcompany, (ii) provide oversight of the usage of the system, or (iii)provide oversight of products produced by the system. The user of thesystem and end user of products produced by the system may be the sameor different companies or even the manufacturer of the system. Byoversight, it is meant that the regulatory body may inspect (i) thesystem, (ii) usage of the system, or (iii) products produced by thesystem in a manner that protects workers operating the system or endusers of the products produced by the system. As part of the oversight,the regulatory body may require validation of operation or end productsof the system and, in turn, provide approval of the system based on thevalidation data provided by the manufacturer, user, or recipients ofproducts of the system.

An example of a business model in which one or more companies operateunder the auspices of a regulatory body is in the case of pharmaceuticalmaterial production. The regulatory body in this case is the Food andDrug Administration 1820. In this business model 1800, the FDA providesoversight to a manufacturer 1805 of a system for producingmacromolecules and/or providing electrophoresis analysis of samples ofthe produced macromolecules. The FDA 1820 also oversees operation of thesystems as used by a macromolecule producer 1810 (hereafter referred toas the producer 1810). Still further, the FDA 1820 oversees use of themacromolecules produced by the system 1500 by a pharmaceuticalresearcher/developer 1815 (hereafter referred to as researcher 1815).

In a typical business cycle, the manufacturer 1805 distributes systems(step 1830) to the producer 1810. The producer 1810 distributesmaterials (step 1835) produced through the use of the system to theresearcher 1815. Prior to distribution of the systems and operation ofthe systems, the manufacturer 1805 may engage in discussions with theresearcher 1815 (step 1825) to assess the needs of the researcher 1815,such as quality of the macromolecules, volume requirements for themacromolecules, and other production needs so as to design the systemwith those needs in mind to ensure commercialization of the systems.

Also included in the business model 1800 are validation and approvalcycles (steps 1840, 1845) by each of the aforementioned companies. Thevalidation and approval cycles 1840, 1845 may be required of each of thecompanies 1805, 1810, and 1815 for each of their respective parts in thebusiness model 1800. For example, in the case of the macromoleculesystem 1600, before a system can be shipped by the manufacturer 1805,the FDA 1820 may require the manufacturer 1805 to participate invalidation 1840 a of the system (e.g., witness and verify data producedby the system, test results, or performance in response to test vectorsprovided to the system). After evaluation of validation data by the FDA1820, the FDA 1820 may grant approval 1845 a of the system. Followingapproval, the distribution of the system (step 1830) can occur, andshipment of the system may follow from the manufacturer 1805 to theproducer 1810.

Similarly, the producer 1810 may have to provide data in the form ofvalidation data to the FDA 1820 for approval 1845 b before the systemcan be used by the producer 1810 to generate macromolecules, forexample. The producer 1810 may have additional requirements for gainingapproval 1845 b from the FDA 1820. For example, the producer 1810 mayhave to customize operational input, as discussed above, to operate thesystem, and test results may have to be shown. In addition, actualmacromolecules produced by the system may also have to be validated bythe producer and sent to the FDA 1820 for approval to ensure quality ofthe macromolecules.

Similarly, the researcher 1815 may also have to send validation data tothe FDA 1820 for approval 1840 c. Typically, this validation andapproval cycle 1840 c, 1845 c is for a drug or other pharmaceuticalproduct produced by the system.

It should be understood that the regulatory body 1820 may be agovernment or non-government agency. For example, in addition to theFDA, the government agency may be the Department of Defense (DOD) thatmay be involved in the oversight of non-government entities to monitorsystems, such as described above, for use in developing vaccines againsttoxic substances, such as anthrax, smallpox, and so forth.

Continuing to refer to the business model of FIG. 18, there may be abusiness advantage for the manufacturer 1805 to distibute a system 1500that has minimal re-validation and re-approval of the system followingdevelopment of operational input by the producer 1810 for its particularmode of operation. By limiting the amount of re-validation andre-approval of the system by the producer 1810, the producer 1810 ismore likely to have shorter re-validation/re-approval cycles by the FDA1820, which, ultimately, may lead to increased profits for the producer1810 due to higher system usage and more distribution of systems by themanufacturer 1805 for this reason.

As discussed above in reference to FIGS. 16 and 17, one way to minimizeexposure of the producer 1810 to re-validation/re-approval cycles 1840,1845 is to provide executable instructions in the system 1500 that areunchangeable by the producer 1810. One way to make the executableinstructions unchangeable is to provide it in a compiled form referredto hereafter as “compiled software” and deploy it in the system 1500,for example, in the form of software or firmware. The compiled softwarepreferably conforms to a known industry standard, such as ANSIprogramming languages or standard protocols for interfacing with devicesor subsystems used to operate the system 1600 (FIG. 16).

A generalized flow diagram of the process just discussed is depicted inFIG. 19. In FIG. 19, a process 1900 is performed by the manufacturer ofthe system 1510. The process 1900 begins (step 1905) upon installationof compiled software in the system 1500. The manufacturer 1805 validatesthe compiled software in the system 1500 (step 1910), which requiresapproval by a regulatory body, such as the FDA 1820. The manufacturer1805 obtains approval of the system 1500 by the regulatory body 1820(step 1915) independent of operational input 1615 to the compiledsoftware 1610. The manufacturer 1805 then distributes the approvedsystem to a customer 1810 (step 1920). The process ends (step 1925)following distribution of the system 1500 (step 1830).

The process 1900 may involve additional steps for gaining validation1840 and approval 1845 by the regulatory body 1820. For example,referring to FIG. 20, the manufacturer 1805 may have an internal process2000 for performing the validation (step 1910). The internal process2000 may begin (step 2005) at a point in which an employee of themanufacturer 1805 compiles software conforming to a known industrystandard (step 2010). The employee then downloads the compiled software1610 to the system (step 2015). In parallel, the same or anotheremployee of the manufacturer 1805 may develop an exhaustive set ofsystem-specific test vectors (step 2020) and use these test vectors totest the compiled software 1610 (step 2025). The employee verifies thatthe compiled software 1610 operates the system 1500 as required toreceive the approval from the regulatory body 1820 (step 2030). Duringthe verification (step 2030), the employee collects data for submissionto the regulatory body 1820 (step 2035). The employee submits the datato the regulatory body 1820 for approval (step 2040), which completesthe internal process 2000 (step 2045).

Referring now to FIG. 21, from the point of view of the producer 1810, aseparate process 2100 is conducted in which customization of the system1500 is provided through custom design and use of operational input1615. The process 2100 begins (step 2105) when the producer 1810generates the operational input 1615 (e.g., declarative instructions)(step 2110). The producer 1810 validates the system (step 2115) with theoperational input 1615 without having to re-validate the compiledsoftware 1610 in the system 1500. The producer 1810 then seeks to obtainapproval of the regulatory body 1820 for the system 1500 with theoperational input 1615 (step 2120), which ends the process 2100 (step2125).

The generation (step 2110) and validation (step 2115) of the operationalinput 1615 can include several substeps, which are shown in a process2200 depicted in FIG. 22. The process 2200 begins (step 2205), and theproducer 1810 generates (step 2110) the operational input 1615. Ascientist or other employee of the macromolecule producer 1810determines desired macromolecule characteristics (step 2210). Thescientist or other employee models (step 2215) the liquid mixture 202containing the macromolecule. The scientist or other employee specifies(step 2220) system 1500 operation to yield the desired macromoleculefrom the liquid mixture 202.

Based on the system specifications, the scientist or other employeegenerates at least one operational input file (file 2225). Thevalidation (step 2115) begins following generation of the operationalinput file (step 2225). The scientist or other employee validates thesystem with the operational input file(s) and collects datacorresponding thereto (step 2230). The producer 1810 submits thecollected data for approval to the regulatory body 1820 absent dataspecific to the compiled software 1610 in the system 1500. In otherwords, at this time in the development cycle of the system 1500, thevalidation and approval cycle (steps 1840 b and 1845 b, respectively) donot include testing, data collection, and submission of the datacorresponding to the compiled software 1610 because the compiledsoftware 1610 has not changed in form or function since gaining approval1845 a by the manufacturer 1805 prior to the distribution 1830 of thesystem 1500. The process 2200 ends (step 2240), and the producer 1810awaits approval 1845 b from the regulatory body 1820.

For any number of reasons, the macromolecule producer 1810 may want toimprove or modify the software in some way to improve the processprovided by the system 1500 for either producing the macromolecules fromthe liquid mixture 202 or performing the electrophoresis analysis by thesystem 1500. In this case, all that the producer 1810 need modify is theoperational input 1615 provided to the compiled software 1610 in thecontrollers 701/1330. In such a case, the producer 1810 can execute adifferent business process that is a subset of the business processesdiscussed above in reference to FIGS. 18-22.

Referring now to FIG. 23, a business process 2300 executed by theproducer 1810 begins (step 2305) upon a decision to change the processexecuted by the system 1600 for any number of reasons. A scientist oremployee of the producer 1810 modifies the operational input file(s)(step 2310), causing a re-validation 1840 b and re-approval 1845 b ofthe system 1600 to be required by the regulatory body 1820. There-validation is performed with the new file(s) and the employeescollect data based on the operation of the system with the newoperational input 1615 (step 2315). The producer 1810 submits the datato the regulatory body 1820 for re-approval absent data specific to thecompiled software 1610 in the system 1500. Again, because the compiledsoftware 1610 is unchangeable by the producer 1810 following itsoriginal validation and approval (steps 1840 a and 1845 a,respectively), the producer 1810 does not need to repeat these steps.

As used herein, a macromolecule can be a large molecule, typically abiological polymer that can be soluble in the liquid mixture. Amacromolecule can be a protein or peptide, for example, a peptidehormone, an enzyme, an enzyme with an associated cofactor, an antibody,a glycoprotein, and the like. A macromolecule can be other biologicalpolymers, for example, polysaccharides, e.g., starches or sugars,polynucleic acids, e.g., deoxyribonucleic (DNA) or ribonucleic acid(RNA), lipids, glycolipids, and the like. A macromolecule can also beother large molecules of interest, for example steroids, carbohydrates,organometallic complexes such as metalloporphyrins, and the like. Amacromolecule can also be a non-biological molecule or polymer. Amacromolecule can be two or more molecules that are associated throughnoncovalent interactions to form a complex, for example, anantibody-antigen complex, an enzyme-inhibitor complex, a multi-domainprotein where the domains are linked by hydrophobic forces, and thelike. Preferably, a macromolecule can be a biopolymer or otherbiological molecule that is the desired product of a particularbioreactor process. For example, in a bioreactor process designed togrow bacteria genetically engineered to express human insulin, themacromolecule is insulin. The macromolecule can also be a molecule thatcan be indicative of the desired product of a particular bioreactorprocess. Most preferably, a macromolecule is a protein. A macromoleculeis typically between about 1,000 and about 200,000 atomic mass units(AMU) in molecular weight. Macromolecules are typically between about10,000 and about 160,000 AMU.

As used herein, components that are smaller or larger than themacromolecule are those that can be separated from the macromolecule byfiltration. Components that are smaller or larger than the macromoleculetypically have a molecular weight that is greater or lesser than,respectively, the molecular weight of the macromolecule. One skilled inthe art will know, however, that the relation of size to molecularweight for macromolecules and similar components is approximate anddepends on a number of factors, including the actual molecular weight,the conformation of the molecule, whether the molecule is aggregated oragglomerated with other molecules, solvent conditions, ionic strength,filter composition, and the like.

As used herein, rough components can include soluble and insolublecomponents. Insoluble components include cells, fragments of cells,non-cellular tissue fragments, insoluble agglomerations ofmacromolecules, particulate contaminants, and the like. Soluble roughcomponents include smaller fragments of cells, macromolecules that arelarger than the macromolecule or are greater in molecular weight thanthe molecular weight of the macromolecule, and the like.

As used herein, fine components include soluble components. Soluble finecomponents include macromolecules that are smaller than themacromolecule or are lesser in molecular weight than the molecularweight of the macromolecule. Also included are small organic andinorganic molecules, for example, salts, amino acids, nucleic acids,cofactors, nutrients, metabolites, other macromolecules, fragments ofthe macromolecule, other biomolecules, and the like.

As used herein, salt components include salts formed from cations suchas sodium, potassium, lithium, cesium, magnesium, manganese, copper,zinc, calcium, iron, ammonium, alkylammonium, phosphonium, sulfonium,and the like. Salt components also include anions including halides,sulfates, thiosulfates, sulfonates, sulfites, nitrates, nitrites,carboxylates, phosphates, phosphates, phosphonates, carbonates,hydroxides, and the like.

The liquid in the liquid mixture containing the macromolecule can be anysolvent, for example, water, organic solvents such as alcohols, e.g.,methanol, ethanol, isopropanol, t-butanol, and the like; ethers, e.g.,dimethyl ether, diethyl ether, tetrahydrofuran, and the like; ketones,e.g., acetone, methyl ethyl ketone, and the like; aromatic solvents,e.g., benzene, toluene, and the like; halogenated solvents, e.g.,chloroform, carbon tetrachloride, trichloroethylene, and the like; polaraprotic solvents, e.g., dimethyl sulfoxide, nitrobenzene, dimethylformamide, n-methylpyrrolidone, acetonitrile, and the like; mixturesthereof, and the like. Typically, the liquid can be water, optionallywith small amounts of one or more organic solvents that are misciblewith water, e.g., ethanol, isopropanol, acetonitrile, and the like.

As used herein, denaturation means changing the conformation and/or thesolubility of a macromolecule to prepare it for analysis. For example,when macromolecule 104 in FIG. 4 is a protein, denaturation can includetransformation from a packed three-dimensional conformation 104 to alinear conformation 104′. Denaturation can also include solubilizing themacromolecule with the denaturing detergent 220. Denaturation can beaccomplished by techniques well known to one skilled in the art, forexample, addition of one or more denaturation agents, application ofheat, disulfide bond reduction, or a combination thereof. Denaturationagents for proteins can include, for example, chaotropic agents e.g.,urea, guanidine hydrochloride, and the like; detergents, e.g. sodiumdodecyl sulfate, potassium laurel sulfate, and the like; disulfidecleavage agents, e.g. dithiothreitol, dithioerythritol, and the like;acids or bases, e.g., trichloroacetic acid, sodium hydroxide, and thelike; and other agents known to the art. Denaturation agents forpolynucleic acids can include, for example, chelation agents, e.g.ethylenediamine tetraaceticacid and the like.

As used herein, a denaturation vessel can be any chamber or conduitwhere denaturation takes place, typically a small volume metal vessel,e.g., a stainless steel vessel between about 1 to about 100 mL. Adenaturation vessel is typically coupled to a heating element, i.e., anydevice known to the art that can be used to heat the fluid mixture, forexample, a resistive heating coil, a microwave heater, a combustionheater such as a gas flame, a heat pump, and the like. A denaturationvessel can also be coupled with a cooling element, for example, a heatpump, refrigeration unit, thermoelectric cooling element, radiator,water cooling coil, and the like. One skilled in the art will recognizethat heating and cooling elements can be part of a single heat exchangerunit.

As used herein, a hydraulic system can be a collection of hydraulicconduits, one or more valves, and one or more pumps, coupled so that thepumps can be used to generate fluid pressure in the hydraulic lines andthe valves can be controlled to direct the pressurized fluid through thelines. A pump can be any device known to the art that can be used togenerate fluid flow, for example, an electro-kinetic pump, or amechanical pump including a peristaltic pump, a syringe pump, animpeller pump, a pneumatic pump, and the like. A valve can be any deviceknown to the art that can be used to control fluid flow, e.g., a needlevalve, a gate valve, a butterfly valve, and the like. An automatedcontroller can be a processor, e.g., an embedded processor, a desktopcomputer, and the like, that can be programmed to control a systemadapted for automatic control, e.g., the hydraulic system.

As used herein, an ion concentration sensor can be any ion concentrationsensor known to one skilled in the art, for example a general ion sensorsuch as a conductance sensor, or a specific ion sensor such as achloride sensor, a hydrogen ion sensor (i.e., a pH sensor), and thelike.

As used herein, a buffer can be any liquid that can be added to themixture to maintain or change the concentration of a particularcomponent, or to combine an additive to change the properties of theprocess. For example, an ionic buffer, e.g., a pH buffer, can change ormaintain the pH of the liquid mixture; a denaturation buffer can containa denaturation agent; a desalination buffer can be a liquidsubstantially free of salts or substantially free of a particular salt,e.g., sodium chloride; a lysis buffer can be a liquid that contains alysing agent (e.g., a detergent) or can be sufficiently low in ionicstrength to lyse cells by ionic shock; and the like. Lysing agents caninclude enzymes, e.g., L-lysine decarboxylase, lysostaphin, lysozyme,lyticase, mutanolysin, and the like. Lysing agents can includedetergents, e.g. glycocholic acid sodium salt hydrate, lithium dodecylsulfate, sodium cholate hydrate, sodium dodecyl sulfate,hexadecyltrimethylammonium bromide, N-Nonanoyl-N-methylglucamine,octyl-b-D-1-thioglucopyranoside,3-(N,N-dimethyloctadecylammonio)propanesulfonate, and the like.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

The following applications contain related subject matter and areincorporated herein by reference in their entirety; U.S. applicationSer. No. 10/601,096 filed on Jun. 20, 2003, entitled “Method forDetection of Molecular species in a Crude Sample Using CapillaryElectrophoresis,” by Shin-Fuw Lin, et al., U.S. application Ser. No.10/601,277 filed on Jun. 20, 2003, entitled “Automated MacromoleculeSample Preparation System,” by George E. Barringer, Jr.; U.S.application Ser. No. 10/600,177 filed on Jun. 20, 2003, entitled “Methodand Apparatus for Operating an Automated Biomolecular PreparationSystem,” by George E. Barringer, Jr., et al.; and U.S. application Ser.No. 10/601,083 filed on Jun. 20, 2003, entitled “Fluid Interface forBioprocessor Systems,” by George E. Barringer, Jr.

1. An apparatus for capillary electrophoresis, comprising: an inletchamber; a capillary electrophoresis column, having a length of at leastabout 20 centimeters, one end of the column being fixed at the interiorof the inlet chamber; and a liquid source adapted for automatic control,that supplies a liquid sample through an input valve into the inletchamber, the sample supplied to be in fluid communication with the endof the column.
 2. The apparatus of claim 1, further comprising an outletvalve located at the inlet chamber.
 3. The apparatus of claim 1, whereinthe sample source pressurizes the inlet chamber to create a pressuredifferential across the length of the column.
 4. The apparatus of claim1, further comprising an outlet chamber, the other end of the columnbeing fixed at the interior of the outlet chamber.
 5. The apparatus ofclaim 4, wherein the sample source pressurizes one chamber compared tothe other chamber to create a pressure differential across the length ofthe column.
 6. The apparatus of claim 5, the liquid source furthercomprising a reservoir supplying a cleaning solution.
 7. The apparatusof claim 6, the liquid source further comprising a mechanical pump. 8.The apparatus of claim 4, further comprising an output valve at eachchamber that is controlled to independently remove liquid from eachchamber.
 9. The apparatus of claim 8, further comprising at least onereservoir supplying a buffer, the liquid source independently supplyingthe buffer to the chambers.
 10. The apparatus of claim 9, furthercomprising a fluid level sensor at each chamber.
 11. The apparatus ofclaim 10, further comprising a filter to separate at least a portion ofinsoluble components from the liquid sample, the liquid source applyingthe liquid to the filter with a pressure differential across the filter.12. The apparatus of claim 4, further comprising electrophoresiselectrodes coupled to an automatically controlled power supply.
 13. Theapparatus of claim 12, further comprising a heat exchanger in thermalcontact with the column.
 14. The apparatus of claim 13, furthercomprising a degas unit that removes at least a portion of gas dissolvedin the liquid.
 15. The apparatus of claim 14, further comprising anautomated detector that detects a molecular analyte in the liquid. 16.The apparatus of claim 15, wherein the detector is located at thecolumn.
 17. The apparatus of claim 16, further comprising an automatedcontroller.
 18. An apparatus for capillary electrophoresis, comprising:a hydraulic system adapted for control by an automated controller,comprising a pump and one or more valves; a rough filter selected toseparate from a macromolecule in a liquid mixture, at least a portion ofone or more rough components in the mixture that are larger than themacromolecule; a fine filter selected to separate from themacromolecule, at least a portion of one or more fine components in themixture that are smaller than the macromolecule; an inlet chamber thatreceives a liquid sample filtered by the rough and fine filters, thesample comprising the macromolecule; a capillary electrophoresis column,having a length of at least about 20 centimeters, one end of the columnbeing fixed at the interior of the inlet chamber; and the hydraulicsystem being controlled to create the liquid sample, the samplecomprising the macromolecule, by applying the liquid mixture to eachfilter, with a pressure differential across each filter, and to supplythe liquid sample to the inlet chamber.
 19. An apparatus for capillaryelectrophoresis, comprising: a hydraulic system adapted for control byan automated controller, comprising a pump and one or more valves; afilter selected to separate, at least in part, a macromolecule in aliquid mixture from one or more salt components in the mixture; an inletchamber that receives a liquid sample, the sample comprising themacromolecule separated from the salt components; a capillaryelectrophoresis column, having a length of at least about 20centimeters, one end of the column being fixed at the interior of theinlet chamber; and an automated controller that controls the hydraulicsystem to create the liquid sample, the sample comprising themacromolecule, by applying the liquid mixture to the filter, with apressure differential across the filter, and to supply the sample to theinlet chamber.
 20. An apparatus for capillary electrophoresis,comprising: a hydraulic system adapted for control by an automatedcontroller, comprising a pump and one or more valves; a lysis unit thatlyses cells in a liquid mixture comprising cells and a macromolecule; afilter selected to separate from the macromolecule, at least a portionof components in the mixture that are larger than the macromolecule, thecomponents comprising insoluble lysed cell components; an inlet chamberthat receives a liquid sample, the sample comprising a macromoleculeseparated from the insoluble lysed cell components; a capillaryelectrophoresis column, having a length of at least about 20centimeters, one end of the column being fixed at the interior of theinlet chamber; and an automated controller that controls the hydraulicsystem to create the liquid sample, the sample comprising themacromolecule, by applying the liquid mixture to the filter, with apressure differential across the filter, and to supply the sample to theinlet chamber.
 21. An apparatus for capillary electrophoresis,comprising: an inlet chamber and an outlet chamber, the chambers eachcomprising an inlet valve, an output valve, a fluid level sensor, and anelectrode, the electrodes coupled to a power supply; a capillaryelectrophoresis column, having a length of at least about 20centimeters, the opposite ends of the column being fixed at the interiorof the respective chambers; a liquid source comprising a pump and atleast one valved reservoir supplying a buffer, the liquid source beingcoupled to the input valves; and an automated controller that controls:the liquid source and at least one valve to create a pressuredifferential across the length of the column by pressurizing ordepressurizing at least one chamber; the liquid source, the outputvalves, the valved reservoir, and the level sensors to independently:drain the chambers; supply the chambers with liquid to place the liquidin fluid communication with the end of the column in each chamber,including supplying: the buffer to the outlet chamber; and independentlyto the inlet chamber; the buffer and a liquid sample, the liquid samplecomprising a macromolecule; and a power supply to apply a voltagedifferential across the column to cause electrophoresis of themacromolecule in the column.
 22. The apparatus of claim 21, furthercomprising an automated detector that detects the macromolecule.
 23. Theapparatus of claim 22, the liquid source further comprising: a hydraulicsystem adapted for control by an automated controller, comprising a pumpand one or more valves; a rough filter selected to separate from themacromolecule in a liquid mixture comprising the macromolecule, at leasta portion of one or more rough components in the mixture that are largerthan the macromolecule; a fine filter selected to separate from themacromolecule from the macromolecule in a liquid mixture comprising themacromolecule, at least a portion of one or more fine components in themixture that are smaller than the macromolecule; and the hydraulicsystem being controlled to create the liquid sample, the liquid samplecomprising the macromolecule, by applying the liquid mixture to eachfilter, with a pressure differential across each filter.
 24. A methodfor capillary electrophoresis, comprising automatically supplying aliquid sample through a valve to an inlet chamber to place the sample influid communication with a capillary electrophoresis column, the chamberhaving one end of the column fixed at the interior of the chamber, andthe column having a length of at least about 20 centimeters.
 25. Themethod of claim 24, further comprising pressurizing the inlet chamber tocreate a pressure differential across the length of the column.
 26. Themethod of claim 24, the other end of the column being fixed at theinterior of an outlet chamber, further comprising directing fluidthrough the column by creating a pressure differential between thechambers.
 27. The method of claim 26, further comprising creating apressure differential by electro-kinetic pumping.
 28. The method ofclaim 26, further comprising creating a pressure differential bymechanical pumping.
 29. The method of claim 26, further comprisingindependently directing liquid from each chamber to a waste site. 30.The method of claim 29, further comprising independently supplying abuffer to each chamber.
 31. The method of claim 30, furtherindependently sensing the fluid level in at least one chamber.
 32. Themethod of claim 31, further comprising separating at least a portion ofinsoluble components from the liquid sample by applying the liquid to afilter with a pressure differential across the filter.
 33. The method ofclaim 26, further comprising applying a voltage differential across thecolumn to create electrophoretic flow in the column.
 34. The method ofclaim 33, further comprising cooling the column.
 35. The method of claim34, further comprising degassing at least a portion of gas dissolved inthe liquid sample and the buffer.
 36. The method of claim 26, furthercomprising detecting a molecular analyte in the liquid sample.
 37. Themethod of claim 36, the molecular analyte being a macromolecule.
 38. Amethod for capillary electrophoresis comprising automatically: acquiringa liquid mixture, the mixture comprising a macromolecule, one or morerough components that are larger than the macromolecule, and one or morefine components that are smaller than the macromolecule; creating aliquid sample, the sample comprising the macromolecule, by separatingfrom the macromolecule at least a portion of the components by applyingthe mixture to each of a plurality of filters, with a pressuredifferential across each filter, the filters comprising a rough filterselected to separate at least a portion of the rough components and afine filter selected to separate at least a portion of the finecomponents; and supplying the liquid sample through a valve to an inletchamber to place the sample in fluid communication with a capillaryelectrophoresis column, the chamber having one end of the column fixedat the interior of the chamber, and the column having a length of atleast about 20 centimeters.
 39. A method for capillary electrophoresis,comprising automatically: acquiring a liquid mixture, the mixturecomprising a macromolecule and one or more salt components; creating aliquid sample, the sample comprising the macromolecule, by separatingthe macromolecule from at least a portion of the salt components, byapplying the mixture to a filter with a pressure differential across thefilter and supplying the liquid sample through a valve to an inletchamber to place the sample in fluid communication with a capillaryelectrophoresis column, the chamber having one end of the column fixedat the interior of the chamber, and the column having a length of atleast about 20 centimeters.
 40. A method for capillary electrophoresiscomprising automatically: acquiring a liquid mixture, the mixturecomprising a macromolecule and one or more cells; lysing at least aportion of the cells; and creating a liquid sample, the samplecomprising the macromolecule, by separating from the macromolecule atleast a portion of components larger than the macromolecule, thecomponents comprising insoluble lysed cell components, by applying themixture to a filter with a pressure differential across the filter; andsupplying the liquid sample through a valve to an inlet chamber to placethe sample in fluid communication with a capillary electrophoresiscolumn, the chamber having one end of the column fixed at the interiorof the chamber, and the column having a length of at least about 20centimeters.
 41. A method for capillary electrophoresis, comprisingautomatically: supplying a liquid sample through a valve to an inletchamber to place the sample in fluid communication with a capillaryelectrophoresis column: the chamber having one end of the column fixedat the interior of the chamber; the column having a length of at leastabout 20 centimeters; and the sample comprising a macromolecule;directing fluid through the column by creating a pressure differentialbetween the inlet chamber and an outlet chamber, the other end of thecolumn being fixed at the interior of the outlet chamber; independentlycontrolling the fluid lever in each chamber by: sensing the fluid levelin each chamber; supplying a buffer to each chamber; and directingliquid from each chamber to a waste site; and electrophoreticallyseparating the macromolecule in the column by applying a voltagedifferential across the column.
 42. The method of claim 41, furthercomprising detecting the macromolecule.
 43. The method of claim 42,further comprising: acquiring a liquid mixture, the mixture comprisingthe macromolecule, one or more rough components that are larger than themacromolecule, and one or more fine components that are smaller than themacromolecule; creating the liquid sample, by separating from themacromolecule at least a portion of the components by applying themixture to each of a plurality of filters, with a pressure differentialacross each filter, the filters comprising a rough filter selected toseparate at least a portion of the rough components and a fine filterselected to separate at least a portion of the fine components.
 44. Anapparatus for capillary electrophoresis, comprising: means forautomatically supplying a liquid sample through a valve to an inletchamber to place the sample in fluid communication with a capillaryelectrophoresis column, the chamber having one end of the column fixedat the interior of the chamber, and the column having a length of atleast about 20 centimeters; and means for causing electrophoresis in thecolumn.
 45. An apparatus for electrophoretic separation of amacromolecule, comprising: a plurality of valves; a rough separationcircuit comprising a rough pump, a first stage rough filter selected toseparate rough components, and a second stage rough filter selected toseparate rough components that pass through the first stage roughfilter; a fine/desalination circuit, comprising a fine pump, a reservoirthat supplies a desalination buffer, and a fine filter s selected toseparate fine components from the macromolecule; a denaturation circuitcomprising a denaturation pump, a denaturing vessel comprising a heatingelement and a cooling element, a precipitation pump, a reservoirsupplying a denaturation buffer, a reservoir supplying a pH buffer, a pHsensor and a precipitation filter selected to separate insolubledenaturation precipitate components; a capillary electrophoresis circuitcomprising an inlet chamber and an outlet chamber, the chambers eachcomprising an inlet valve, an output valve, a fluid level sensor, and anelectrode, the electrodes coupled to a power supply; a capillaryelectrophoresis column, having a length of at least about 20centimeters, the opposite ends of the column being fixed at the interiorof the respective chambers; a buffer reservoir supplying the chambers;an automated controller in electronic communication with the pumps, theelements, the valves, the sensors and the power supply that controls theapparatus to: acquire a liquid mixture from a sampling site, the mixturecomprising a macromolecule, rough components, and fine components;separate at least a portion of rough components from the macromoleculein the rough separation circuit; separate at least a portion of finecomponents from the macromolecule in the fine/desalination separationcircuit, the fine components comprising salt components; denature themacromolecule in the denaturation circuit; and electrophoreticallyseparate the denatured macromolecule from other components by employingthe capillary electrophoresis circuit.